抗寄生虫药物双氯酚对金黄色葡萄球菌的体外和体内抗菌活性研究

In vitro and in vivo antimicrobial activity of the antiparasitic drug dichlorophen against Staphylococcus aureus

摘要:

目的 研究抗寄生虫药物双氯酚(DIC)对金黄色葡萄球菌的抗菌活性。方法 通过微量肉汤稀释试验和纸片扩散试验检测金黄色葡萄球菌对DIC的敏感性和耐药诱导能力,时间-杀菌曲线检测DIC的杀菌效率,结晶紫和XTT染色检测DIC对金黄色葡萄球菌生物膜形成的抑制作用和对已形成生物膜的清除作用,Cell Coun-ting Kit-8(CCK-8)试剂盒检测DIC的细胞毒性,构建皮肤脓肿感染的小鼠模型检测DIC的体内抗菌活性和体内毒性。结果 DIC对金黄色葡萄球菌标准株的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为2~4 μg/mL、2~8 μg/mL;对金黄色葡萄球菌临床菌株的MIC和MBC分别为2~8 μg/mL、8~32 μg/mL。纸片扩散试验中,DIC对金黄色葡萄球菌标准株具有明显的浓度依赖抑菌活性。时间-杀菌曲线显示,DIC浓度为4×MIC时,处理2 h金黄色葡萄球菌标准株ATCC 29213和处理4 h金黄色葡萄球菌ATCC 43300可分别将活菌浓度从(5.51±0.27) Log₁₀ CFU/mL、(5.44±0.08)Log₁₀ CFU/mL降低至最低检测限。亚抑菌浓度的DIC连续作用于细菌传代15次后,未见金黄色葡萄球菌耐药突变株形成。2 μg/mL的DIC可显著抑制金黄色葡萄球菌生物膜的形成,使其生物膜的总量从(100±7.49)%减少至(11.12±2.86)%(P<0.001);2 μg/mL的DIC可显著清除已形成的金黄色葡萄球菌生物膜,使生物膜的总量从(100±10.34)%减少至(42.53±16.87)%(P<0.001)。DIC能显著降低小鼠脓肿组织中的金黄色葡萄球菌活菌载量, 使活菌数从(9.54±0.46)Log₁₀ CFU/脓肿降低至(7.78±0.62)Log₁₀ CFU/脓肿 (P<0.05)。苏木精-伊红染色结果显示,DIC能明显缩小小鼠脓肿面积并降低组织中的炎性细胞浸润,具有良好的体内耐受性。结论 DIC的细胞毒性小且体外和体内抗菌活性显著,有望成为耐药金黄色葡萄球菌感染的替代治疗方案。

Abstract:

Objective To explore the antimicrobial activity of antiparasitic drug dichlorophenol (DIC)against Staphylococcus aureus (S. aureus). Methods Antimicrobial susceptibility and resistance inducing ability of S. aureus against DIC was detected by micro-broth dilution assay and disc diffusion test; bactericidal efficacy of DIC was assessed by time-killing curve; inhibitory effect of DIC on the formation of biofilm and eradicating of formed biofilm of S. aureus was detected by crystal violet and XTT staining; cytotoxicity of DIC was detected by cell counting kit-8 (CCK-8), and a mouse model of skin abscess infection was constructed to detect the in vivo antimicrobial activity and toxicity of DIC. Results The minimal inhibitory (MIC) and minimal bactericidal concentration (MBC) of DIC against S. aureus standard strains were 2-4 μg/mL and 2-8 μg/mL, respectively. The MIC and MBC of DIC against S. aureus clinical strains were 2-8 μg/mL and 8-32 μg/mL, respectively. Disc diffusion test indicated the obvious concentration-dependent bacterial growth inhibitory effects of DIC on S. aureus standard strains. Time-killing assay revealed that DIC concentration of 4×MIC was found to reduce the viable bacterial cells of S. aureus standard strains ATCC 29213 and ATCC 43300 from (5.51±0.27) Log₁₀ CFU/mL and (5.44±0.08) Log₁₀ CFU/mL to the limit of detection after 2 hours and 4 hours treatment, respectively. No drug-resistant mutant strains of S. aureus were found after 15 consecutive passage of DIC with subinhibitory concentration on bacteria. 2 μg/mL DIC could significantly inhibit the formation of S. aureus biofilm and reduce the total amount of biofilm from (100±7.49)% to (11.12±2.86)% (P<0.001). 2 μg/mL DIC significantly eradicated the formed S. aureus biofilm and reduced the total biofilm from (100±10.34)% to (42.53±16.87)% (P<0.001). DIC could significantly reduce the viable bacterial load of S. aureus in abscess in mice, and reduce the number of viable bacteria from (9.54±0.46) Log₁₀ CFU/abscess to (7.78±0.62) Log₁₀ CFU/abscess (P<0.05). Hematoxylin-eosin staining result showed that DIC could significantly reduce the abscess area and inflammatory cell infiltration in mice tissue, and was well tolerated in vivo. Conclusion DIC has low cytotoxicity and obvious in vitro and in vivo antimicrobial activity, which is expected to be an alternative treatment for drug-resistant S. aureus infection.